The efficient and rapid purification of biological materials such as proteins, nucleic acids or polysaccharides, has been of great interest. Immunoaffinity chromatography is advantageous since, under the proper conditions, purification of 1,000-10,000 fold can be attained. Cf. Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).
One protein for which a rapid and efficient purification system is desirable is the biological modifier Mullerian Inhibiting Substance (MIS) produced in vivo by the ovary and testis. MIS is a testicular protein responsible for regression of the anlagen of the female reproductive tract in the fetal urogenital ridge, as well as other differentiative functions such as inhibition of oocyte meiosis and lung surfactant. Previous studies in A431 cells and more recently in fetal lung suggest that MIS is a potent inhibitor of epidermal growth factor receptor autophosphorylation. Cf. Cigarroa et al., Growth Factors 1:79-91 (1989); Coughlin et al., Mol. and Cell. Endocrinol. 49:75-86 (1985); and Catlin et al., Metabolism.
Human Mullerian Inhibiting Substance has been cloned and expressed recombinantly (rhMIS) in animal cells as a 140 kDa homodimer (Cate et al., Cell 45:685 (1986)). By virtue of carboxy-terminal amino acid homology, MIS is a member of a large gene family that includes TGF-.beta. (Derynck et al., Nature 316:701-5 (1985)), inhibin (Mason et al., Nature 318:659-63 (1985)), activin (Ling et al., Nature 321:779-82 (1986)), Vg1 from Xenopus (Weeks et al., Cell 51:861-7 (1987)), the Drosophila decapentaplegia protein (Padgett et al., Nature 325:81-4 (1987)), and the bone morphogenesis factors (Wozney et al., Science 242:1528-34 (1988)).
RhMIS is a 140 kDa glycoprotein composed of two identical subunits which, under disulfide bond reducing conditions, migrates on polyacrylamide gel electrophoresis at an apparent molecular weight of 70 kDa. The protein can be proteolytically cleaved in approximately one hour by exogenous plasmin into two distinct fragments that migrate electrophoretically as 57 kDa and 12.5 kDa moieties with cleavage at residue 427 of the intact 535 amino acid monomer as demonstrated by Pepinsky (Pepinsky et al., J. Biol. Chem. 263(35):18961-4 (1988)). Prolonged exposure to plasmin can result in cleavage of MIS at additional sites. In addition, purification of MIS by known techniques can be contaminated by other proteases that also cleave MIS.
MIS has been proposed as a potential growth inhibitor of epithelial human tumors of Mullerian origin such as endometrial, Fallopian tubal, cervical, and certain ovarian neoplasms. Experimental evidence using purified bovine MIS support this hypothesis (U.S. patent application Ser. No. 06/792,233, filed Oct. 19, 1985, now U.S. Pat. No. 5,011,687; Fuller et al., J. Clin, Endocrinol. Metab. 54:1051-5 (1982); Fuller et al., Gynecol. Oncol. 22:135-48 (1985)). purified rhMIS used in similar in vitro assays, however, demonstrate limited anti-cancer activity (Wallen et al., Cancer Res. 49:2005-11 (1989)),
Mullerian Inhibiting Substance may be obtained by a variety of different methods. U.S. patent application Ser. No. 06/792,233, filed Oct. 19, 1985, now U.S. Pat. No. 5,011,687, and entitled "Purified Mullerian Inhibiting Substance and Process for Treating Human Ovarian Cancer Cells," describes a process for purifying MIS from testes by using aqueous polar dissociative solutions, separation of DNA and RNA, fractionation by gel filtration chromatography, and isolation of the MIS. U.S. Pat. No. 4,404,188, filed Jul. 29, 1981 and entitled "Purified Mullerian Inhibiting Substance and Method of Purification" describes a process for purifying MIS from testes which comprises treatment with a protease inhibitor, chromatography on ion exchange, chromatography on wheat germ lectin, on concanavalin A and/or on a supported triazinyl dye. U.S. Pat. No. 4,487,833, filed on Mar. 1, 1982 and entitled "Method of Preparing Hybridomas and of Purifying Immunogenic Materials" describes a process for separating MIS using immunoaffinity chromatography. MIS has also been obtained from recombinant DNA techniques as disclosed by Cate (Cate, et al., Cell 45:685-698 (1986)). None of the references, however, describe a method of recovering substantially pure MIS which retains an antiproliferative activity or is essentially free of enzymes having proteolytic activity against MIS.
A need, therefore, continues to exist for the development of highly efficient techniques for immunoaffinity chromatography, wherein a biological substance can be isolated from a complex biological mixture without interference from contaminating proteolytic enzymes or other factors that impede MIS antiproliferative activity. In particular, the need exists for an immunoaffinity chromatography method which can isolate and purify MIS so that the isolate will be essentially free from undesired proteolysis or other factors which may alter MIS activity.